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L D Kohn

Publications and source records attributed to L D Kohn.

At least 55 records · Page 3Linked to original sources
PubMed1999

Graves' immunoglobulins activate phospholipase A2 by recognizing specific epitopes on thyrotropin receptor.

Thyroid-stimulating IgG from Graves' patients bind to the TSH receptor and activate both adenylyl cyclase (AC) and phospholipase A2 (PLA2) in FRTL5 thyroid cells. Both activities have been associated with increased thyroid cell growth and function; evidence exists that subpopulations of Graves' IgG can stimulate either AC or PLA2 cascades and that the activation of both is associated with the largest goiters in patients. Studies using chimeras of the human TSHR receptor (hTSHR) and the LH-CG receptor show that most patients with Graves' disease have cAMP-stimulating IgG that require epitopes on the N-terminal portion of the TSHR extracellular domain; epitopes associated with PLA2 activation are not clear. To address this question we used stably transfected Chinese hamster ovary (CHO) cells containing the wild-type hTSHR and the hTSHR chimera with residues 8-165 (Mc1+2) substituted by equivalent residues of the LH-CG receptor. PLA2 activity, measured as arachidonic acid (AA) release, was determined in 32 patients with Graves' disease. We show that 72% of Graves' patients have IgG able to stimulate PLA2 in CHO cells transfected with the TSHR and that AA release induced by Graves' IgG was significantly reduced (P = 0.022) in the CHO-Mc1+2-transfected cells (193 +/- 88% vs. 131 +/- 67%, respectively). Unlike IgG, the effect of TSH was not modified in the CHO-Mc1+2-transfected cells. When we compared the AC- and PLA2-stimulating activities of these 32 IgG in wild-type TSHR transfectants, we found that 63% of Graves' patients have antibodies able to stimulate both PLA2 and AC, whereas some patients' IgG were active only in AC or PLA2 assays. Of the patients with IgG having activity in both assays in wild-type TSHR transfectants, 50% of the IgG lost their stimulatory activities in both AA release and cAMP assays in Mc1+2 cells. Of the remainder, some IgG maintained their activity in one (AA release) or the other (cAMP) assay when measured in Mc1+2 chimeras. Thus, our data show that the N-terminal portion of extracellular domain of the TSHR is required for PLA2 as well as AC activation by IgG from patients with Graves' disease. These data also demonstrate that patients with Graves' disease have heterogeneous autoantibodies that selectively activate AC and PLA2 pathways and suggest that patients with autoantibodies active in both assays have more severe disease, with higher thyroid hormone levels and larger goiters.

Adult↗
PubMed1998

Autoregulation of thyroid-specific gene transcription by thyroglobulin.

Thyroglobulin (TG), the primary synthetic product of the thyroid, is the macromolecular precursor of thyroid hormones. TG synthesis, iodination, storage in follicles, and degradation control thyroid hormone formation and secretion into the circulation. Thyrotropin (TSH), via its receptor (TSHR), increases thyroid hormone levels by up-regulating expression of the sodium iodide symporter (NIS), thyroid peroxidase (TPO), and TG genes. TSH does this by modulating the expression and activity of several thyroid-specific transcription factors, thyroid transcription factor (TTF)-1, TTF-2, and Pax-8, which coordinately regulate NIS, TPO, TG, and the TSHR. Major histocompatibility complex class I gene expression, which also is regulated by TTF-1 and Pax-8 in the thyroid, is decreased simultaneously. This helps maintain self-tolerance in the face of TSH-increased gene products necessary for thyroid hormone formation. In this report we show that follicular TG counter-regulates TSH-increased, thyroid-specific gene transcription by suppressing expression of the TTF-1, TTF-2, and Pax-8 genes. This decreases expression of the TG, TPO, NIS, and TSHR genes, but increases class I expression. TG acts transcriptionally, targeting, for example, a sequence within 1.15 kb of the 5' flanking region of TTF-1. TG does not affect ubiquitous transcription factors regulating TG, TPO, NIS, and/or TSHR gene expression. The inhibitory effect of TG on gene expression is not duplicated by thyroid hormones or iodide and may be mediated by a TG-binding protein on the apical membrane. We hypothesize that TG-initiated, transcriptional regulation of thyroid-restricted genes is a normal, feedback, compensatory mechanism that limits follicular function and contributes to follicular heterogeneity.

Animals↗
PubMed1998

Newly synthesized Rho A, not Ras, is isoprenylated and translocated to membranes coincident with progression of the G1 to S phase of growth-stimulated rat FRTL-5 cells.

Ras and Rho are involved in the regulation of signal transduction events governing cell growth and cell proliferation. Protein prenylation is essential for the activation and/or the translocation of these small GTPases; however, protein geranylgeranylation rather than farnesylation is required for G1/S transition. We studied prenylation and translocation of Ras and Rho A during G1/S progression in growth-stimulated rat thyroid FRTL-5 cells. Immunoblot analysis revealed that both Ras and Rho A were detected in membrane fractions at G0. Rho A was eliminated from the membrane fraction during G1 and was not detected on the membrane at mid-G1. Translocation of Rho A from the cytoplasm back to the membranes was observed during late G1 phase. In contrast, Ras remains in the membrane fraction through the cell cycle progression from G1 to S phase. The immunoprecipitation of Rho A from the membrane fraction demonstrated that newly synthesized Rho A, labeled by pulsing cells with [35S]methionine and [35S]cysteine, was geranylgeranylated and associated with the membrane in late G1. These results indicate that Rho A, not Ras, was eliminated from membrane fraction during G1 progression and that newly synthesized Rho A is geranylgeranylated and translocated to membranes during G1/S progression in growth-stimulated FRTL-5 cells.

Animals↗
PubMed1998

Spontaneous autoimmune disease in (NZB x NZW)F1 mice is ameliorated by treatment with methimazole.

(NZB x NZW)F1 mice spontaneously develop with age an autoimmune disease that resembles the human disease, systemic lupus erythematosus (SLE). The present study demonstrates that methimazole (MMI), an agent used in the treatment of autoimmune thyroid disease, is effective in mitigating the development of this SLE-like autoimmune disease in (NZB x NZW)F1 mice. MMI significantly reduces the incidence and severity of proteinuria and deposition of immune complexes in the kidney. Previous studies have demonstrated that development of an experimentally induced SLE, which was prevented by MMI treatment, depended on the expression of MHC class I molecules. We now report that class I levels on both T cells and B cells from old (NZB x NZW)F1 MHC class I are markedly elevated relative to those from young F1 mice. Furthermore, treatment of (NZB x NZW)F1 mice with MMI reduced MHC class I expression on their PBL concomitant with amelioration of disease, raising the possibility that class I molecules may play a role in the generation of spontaneous autoimmune disease in these mice.

Aging↗
PubMed1998

High frequency of T-cell lines responsive to immunodominant epitopes of thyrotropin receptor in healthy subjects.

In this study we analyzed the proliferative response to the extracellular domain of thyrotropin receptor (TSHR-ECD) of T-cell lines raised from healthy subjects. We found high frequencies of cell lines reactive to TSHR-ECD, ranging from 12% to 37%. The response of the cell lines to a set of overlapping peptides of TSHR-ECD showed that the most recognized epitopes by T lymphocytes are on the C-terminal portion. In particular, the regions of residues 360-396 and 258-277 are immunodominant in T-lymphocyte reactivity. A group of cell lines specific for the peptides of TSHR-ECD lost the response to the peptides during time in culture. However, these lines were still responsive to TSHR extracellular domain. The cloning of one of these lines showed three types of T-cell clones: (1) CD4+ clones (n = 4) highly responsive to the TSHR-ECD; (2) CD4+ clones (n = 4) low responsive to TSHR-ECD; (3) CD8+ clones (n = 9) not responsive to TSHR-ECD. The first group of clones was stable during time in culture, while the second group was characterized by the loss of the specific response to TSHR-ECD after some weeks from the first analysis. The observation of a spontaneous anergy in the second group of CD4+ clones suggests that mechanisms of control of the lymphocyte response to TSHR-ECD could be activated in vitro.

Adult↗
PubMed1998

Thyroid transcription factor 1 is calcium modulated and coordinately regulates genes involved in calcium homeostasis in C cells.

Thyroid transcription factor 1 (TTF-1) was identified for its critical role in thyroid-specific gene expression; its level in the thyroid is regulated by thyrotropin-increased cyclic AMP levels. TTF-1 was subsequently found in lung tissue, where it regulates surfactant expression, and in certain neural tissues, where its function is unknown. Ligands or signals regulating TTF-1 levels in lung or neural tissue are unknown. We recently identified TTF-1 in rat parafollicular C cells and parathyroid cells. In this report, we show that TTF-1 is present in the parafollicular C cells of multiple species and that it interacts with specific elements on the 5'-flanking regions of the extracellular Ca2+-sensing receptor (CaSR), calmodulin, and calcitonin genes in C cells. When intracellular Ca2+ levels are increased or decreased in C cells, by the calcium ionophore A23187, by physiologic concentrations of the P2 purinergic receptor ligand ATP, or by changes in extracellular Ca2+ levels, the promoter activity, RNA levels, and binding of TTF-1 to these genes are, respectively, decreased or increased. The changes in TTF-1 inversely alter CaSR gene and calcitonin gene expression. We show, therefore, that TTF-1 is a Ca2+-modulated transcription factor that coordinately regulates the activity of genes critical for Ca2+ homeostasis by parafollicular C cells. We hypothesize that TTF-1 similarly coordinates Ca2+-dependent gene expression in all cells in which TTF-1 and the CaSR are expressed, i. e., parathyroid cells, neural cells in the anterior pituitary or hippocampus, and keratinocytes.

Animals↗
PubMed1998

Regulation of major histocompatibility class II gene expression in FRTL-5 thyrocytes: opposite effects of interferon and methimazole.

Aberrant expression of major histocompatibility complex (MHC) class II antigens is associated with autoimmune thyroid disease; aberrant expression duplicating the autoimmune state can be induced by interferon-gamma (IFNgamma). We have studied IFNgamma-induced human leukocyte antigen (HLA)-DR alpha gene expression in rat FRTL-5 thyroid cells to identify the elements and factors important for aberrant expression. Using an HLA-DR alpha 5'-flanking region construct from -176 to +45 bp coupled to the chloramphenicol acetyltransferase reporter gene, we show that there is no basal class II gene expression in FRTL-5 thyroid cells, that IFNgamma can induce expression, and, as is the case for antigen-presenting cells from the immune system, that IFNgamma-induced expression requires several highly conserved elements on the 5'-flanking region, which, from 5' to 3', are the S, X1, X2, and Y boxes. Methimazole (MMI), a drug used to treat patients with Graves' disease and experimental thyroiditis in rats or mice, can suppress the IFNgamma-induced increase in HLA-DR alpha gene expression as a function of time and concentration; MMI simultaneously decreases IFNgamma-induced endogenous antigen presentation by the cell. Using gel shift assays and the HLA-DR alpha 5'-flanking region from -176 or -137 to +45 bp as radiolabeled probes, we observed the formation of a major protein-DNA complex with extracts from FRTL-5 cells untreated with IFNgamma, termed the basal or constitutive complex, and formation of an additional complex with a slightly faster mobility in extracts from cells treated with IFNgamma. MMI treatment of cells prevents IFNgamma from increasing the formation of this faster migrating complex. Formation of both complexes is specific, as evidenced in competition studies with unlabeled fragments between -137 and -38 bp from the start of transcription; nevertheless, they can be distinguished in such studies. Thus, high concentrations of double stranded oligonucleotides containing the sequence of the Y box, but not S, X1, or X2 box sequences, can prevent formation of the IFNgamma-increased faster migrating complex, but not the basal complex. Both complexes involve multiple proteins and can be distinguished by differences in their protein composition. Thus, using specific antisera, we show that two cAMP response element-binding proteins, activating transcription factor-1 and/or -2, are dominant proteins in the upper or basal complex. The upper or basal complex also includes c-Fos, Fra-2, Ets-2, and Oct-1. A dominant protein that distinguishes the IFNgamma-increased lower complex is CREB-binding protein (CBP), a coactivator of cAMP response element-binding proteins. We, therefore, show that aberrant expression of MHC class II in thyrocytes, induced by IFNgamma, is associated with the induction or increased formation of a novel protein-DNA complex and that its formation as well as aberrant class II expression are suppressed by MMI, a drug used to treat human and experimental autoimmune thyroid disease. Its component proteins differ from those in a major, basal, or constitutive protein-DNA complex formed with the class II 5'-flanking region in cells that are not treated with IFNgamma and that do not express the class II gene.

Animals↗
PubMed1998

Major histocompatibility class II HLA-DR alpha gene expression in thyrocytes: counter regulation by the class II transactivator and the thyroid Y box protein.

Aberrant expression of major histocompatibility complex (MHC) class II proteins on thyrocytes, which is associated with autoimmune thyroid disease, is mimicked by gamma-interferon (gamma-IFN). To define elements and factors that regulate class II gene expression in thyrocytes and that might be involved in aberrant expression, we have studied gamma-IFN-induced HLA-DR alpha gene expression in rat FRTL-5 thyroid cells. The present report shows that class II expression in FRTL-5 thyrocytes is positively regulated by the class II transactivator (CIITA), and that CIITA mimics the action of gamma-IFN. Thus, as is the case for gamma-IFN, several distinct and highly conserved elements on the 5'-flanking region of the HLA-DR alpha gene, the S, X1, X2, and Y boxes between -137 to -65 bp, are required for class II gene expression induced by pCIITA transfection in FRTL-5 thyroid cells. CIITA and gamma-IFN do not cause additive increases in HLA-DR alpha gene expression in FRTL-5 cells, consistent with the possibility that CIITA is an intermediate factor in the gamma-IFN pathway to increased class II gene expression. Additionally, gamma-IFN treatment of FRTL-5 cells induces an endogenous CIITA transcript; pCIITA transfection mimics the ability of gamma-IFN treatment of FRTL-5 thyroid cells to increase the formation of a specific and novel protein/DNA complex containing CBP, a coactivator of CRE binding proteins important for cAMP-induced gene expression; and the action of both gamma-IFN and CIITA to increase class II gene expression and increase complex formation is reduced by cotransfection of a thyroid Y box protein, which suppresses MHC class I gene expression in FRTL-5 thyroid cells and is a homolog of human YB-1, which suppresses MHC class II expression in human glioma cells. We conclude that CIITA and TSH receptor suppressor element binding protein-1 are components of the gamma-IFN-regulated transduction system which, respectively, increase or decrease class II gene expression in thyrocytes and may, therefore, be involved in aberrant class II expression associated with autoimmune thyroid disease.

Animals↗
PubMed1998

The formation of thyrotropin receptor (TSHR) antibodies in a Graves' animal model requires the N-terminal segment of the TSHR extracellular domain.

Immunization of AKR/N mice with murine fibroblasts, transfected with the TSH receptor (TSHR) and a murine major histocompatibility complex class II molecule having the same H-2k haplotype (but not either alone), induces immune thyroid disease with the humoral and histological features of human Graves', including the presence of two different TSHR antibodies (TSHRAbs): stimulating TSHRAbs, which cause hyperthyroidism; and TSH-binding-inhibiting immunoglobulins. The primary functional epitope for both types of antibodies in Graves' patients is on the N-terminal portion of the extracellular domain of the TSHR, residues 25 to 165; most require residues 90-165 to express TSHRAb activity, as evidenced in studies using chimeras of the TSHR and lutropin-choriogonadotropin receptor (LH-CGR). To evaluate the role of this region of the TSHR in the formation of Graves' TSHRAbs, we immunized AKR/N mice with fibroblasts transfected with three human TSHR chimeras with residues 9-165 (Mc1+2), 90-165 (Mc2), or 261-370 (Mc4) substituted by equivalent residues of the rat LH-CGR. Mice immunized with the Mc1+2 and Mc2 chimeras, with the N-terminal portion of the extracellular domain of the TSHR substituted by LH-CGR residues, did not develop TSHRAbs. Mice immunized with the Mc4 chimera, having a major portion of the C-terminal portion of the extracellular domain of the TSHR replaced by comparable LH-CGR residues, can develop TSHRAbs. The results suggest that the N-terminal segment of the TSHR extracellular domain is not only a critical functional epitope for Graves' TSHRAbs, but it is important also in their formation in a mouse model of Graves' disease.

Animals↗
PubMed1998

Regulation of major histocompatibility (MHC) class II human leukocyte antigen-DR alpha gene expression in thyrocytes by single strand binding protein-1, a transcription factor that also regulates thyrotropin receptor and MHC class I gene expression.

The single strand binding protein (SSBP-1) is a positive regulator of TSH receptor gene expression and binds to an element with a GXXXXG motif. The S box of the mouse major histocompatibility class II gene has multiple GXXXXG motifs and can also bind SSBP-1. The S box is one of four highly conserved elements on the 5'-flanking region of class II genes that are necessary for interferon-gamma (IFNgamma) to overcome the normally suppressed state of the gene and induce aberrant class II expression. In this report we show that SSBP-1, when overexpressed in FRTL-5 thyroid cells, is a positive regulator of human leukocyte antigen (HLA)-DR alpha class II gene expression, as is IFNgamma or the class II trans-activator (CIITA). This is evidenced by increased exogenous promoter activity, increased endogenous RNA levels, and increased endogenous antigen expression after transfecting full-length SSBP-1 complementary DNA together with a HLA-DR alpha promoter-reporter gene chimera into TSH-treated FRTL-5 thyroid cells whose endogenous SSBP-1 levels are low. IFNgamma reverses the ability of TSH to decrease endogenous SSBP-1 RNA levels. Also, whereas SSBP-1 transfection does not cause any increase in IFNgamma-induced exogenous promoter activity, transfection of SSBP-1 and CIITA additively increases endogenous class II RNA levels to levels measured in cells treated with IFNgamma. Further, competition studies show that SSBP-1 binding is necessary for formation of the double strand protein/DNA complexes that are seen in electrophoretic mobility shift assays when the class II 5'-flanking region is incubated with extracts from IFNgamma-treated FRTL-5 cells and that have been previously associated with IFNgamma-induced aberrant class II expression. These data suggest that SSBP-1 is involved in the action of IFNgamma to overcome the normally suppressed state of the class II gene; it functions together with CIITA, whose expression is independently increased by IFNgamma. The effect of SSBP-1 as a positive regulator of class II promoter activity is lost in cells maintained without TSH, in which endogenous SSBP-1 RNA levels are already high in the absence of aberrant class II gene expression. These data suggest that high levels of endogenous SSBP-1 are insufficient to cause aberrant class II expression, but, rather, TSH or IFNgamma treatment additionally modulates the cell, albeit differently, such that transfected or endogenous SSBP-1, respectively, can express its positive regulatory activity. The effect of TSH is consistent with reports indicating that TSH enhances the ability of IFNgamma to increase class II gene expression despite the fact IFNgamma increases endogenous SSBP-1 to only the same levels as in cells untreated with TSH. Finally, the effect of SSBP-1 as a positive regulator is lost when GXXXXG motifs, which exist on both the coding and noncoding strands of the S box, are mutated. Consistent with this, mutation and oligonucleotide competition studies show that GXXXXG motifs are necessary for either strand of the S box to bind protein/DNA complexes containing SSBP-1 in FRTL-5 cell extracts or to bind to recombinant SSBP-1. They also suggest that the SSBP-1-binding sites on either strand of the HLA-DR alpha S box are functionally distinct. We conclude from these data that the positive regulatory action of SSBP-1 on class II gene expression involves GXXXXG motifs on each strand of the highly conserved S box of the class II 5'-flanking region. As SSBP-1 is modulated by IFNgamma and is involved in class I and TSH receptor as well as class II gene expression in FRTL-5 cells, the sum of the data supports the hypotheses that common transcription factors regulate all three genes, and their altered activities may contribute to the development of autoimmunity.

Animals↗
PubMed1998

Identification of thyroid transcription factor-1 in C cells and parathyroid cells.

We have identified thyroid transcription factor-1 (TTF-1) mRNA in parafollicular C cells of the adult rat thyroid and in parathyroid cells; in each case the signal is stronger than in the thyrocytes themselves. We additionally identify TTF-1 RNA in other adult rat tissues not previously recognized to contain TTF-1 in developmental or knockout studies: basal layer cells of flattened squamous epithelium in skin and esophagus, three layers of the retina, i.e. pigmented epithelium, inner granular layer, and ganglion cell layer, and three areas of the brain, i.e. anterior pituitary, cerebellum, and hippocampus. Based on the array of cells that are shown to contain TTF-1 in this report, we speculate that TTF-1 may have a role in the regulation of genes important in calcium homeostasis in the intact organism as well as different tissues.

Animals↗
PubMed1998

Thyrotropin receptor epitopes recognized by graves' autoantibodies developing under immunosuppressive therapy.

Abnormal modulation of the immune system is a prerequisite for the expression of Graves' disease. Thus, when hyperthyroidism developed in a renal transplant recipient under long term immunosuppression with cyclosporine A and prednisone, we carefully evaluated the basis for her hyperthyroidism and her state of immunosuppression. Immunosuppression was confirmed by finding markedly deficient lymphocyte responses to common mitogens. Lymphocyte phenotype frequencies were those previously found in Graves', i.e. elevated frequencies of CD3/DR, CD5/26, and CD3/25 lymphocytes. There was also reversal of the CD4/CD8 ratio due to increased CD8 frequency; this is not a typical finding in autoimmune hyperthyroidism, but has been seen in the intrathyroidal lymphocyte populations of some Graves' patients and is associated with other forms of autoimmunity. The patient's serum contained a broad spectrum of TSH receptor autoantibodies (TSHRAbs) characteristic of Graves' disease. To determine whether these were an unusual population of autoantibodies, we determined their functional epitopes before and for nearly 1 yr after radioiodine therapy. Stimulating TSHRAbs that increase cAMP levels were human receptor (TSHR) specific and consistently recognized functional epitopes located on TSHR residues 90-165. Stimulating TSHRAbs that increased arachidonate release and inositol phosphate levels recognized residues 25-90, as did TSH binding inhibitory Igs present in the patient. These data demonstrate that Graves' disease with a wide array of TSHRAbs can develop in a patient despite adequate immunosuppression. More importantly, they show that the cAMP-stimulating TSHRAb associated with disease expression in this patient had a homogeneous subtype dependent on TSHR residues 90-165. As persistence of this type of TSHRAb over time has been associated with resistance to methimazole therapy in Graves' patients, we speculate that the development and persistence of TSHRAb with this homogeneous epitope may be linked to resistance to immunosuppressive therapy.

Animals↗
PubMed1998

Iodide suppression of major histocompatibility class I gene expression in thyroid cells involves enhancer A and the transcription factor NF-kappa B.

High concentrations of iodide can induce transient, clinical improvement in patients with autoimmune Graves' disease. Previous work has related this iodide action to the autoregulatory effect of iodide on the growth and function of the thyroid; more recently, we additionally related this to the ability of iodide to suppress major histocompatibility (MHC) class I RNA levels and antigen expression on thyrocytes. In this report, we describe a transcriptional mechanism involved in iodide suppression of class I gene expression, which is potentially relevant to the autoregulatory action of iodide. Transfection experiments in FRTL-5 cells show that iodide decreases class I promoter activity and that this effect can be ascribed to the ability of iodide to modulate the formation of two specific protein/DNA complexes with enhancer A, -180 to -170 bp, of the class 1 5'-flanking region. Thus, iodide decreases the formation of Mod-1, an enhancer A complex involving the p50 subunit of NF-kappa B and a c-fos family member, fra-2, which was previously shown to be important in the suppression of class I levels by hydrocortisone. Unlike hydrocortisone, iodide also increases the formation of a complex with enhancer A, which we show, in antibody shift experiments, is a heterodimer of the p50 and p65 subunits of NF-kappa B. The changes in these complexes are not duplicated by chloride and are related to the action of iodide on class I RNA levels by the following observations. First, FRTL-5 thyroid cells with an aged phenotype coincidentally lose the ability of iodide to decrease MHC class I RNA levels and to induce changes in either complex. Second, the effect of iodide on class I RNA levels and on enhancer A complex formation with Mod-1 and the p50/p65 heterodimer is inhibited by agents that block the inositol phosphate, Ca++, phospholipase A2, arachidonate signal transduction pathway: acetylsalicylate, indomethacin, and 5,8,11,14-eicosatetraynoic acid. Interestingly, iodide can also decrease formation of the Mod-1 complex and increase formation of the complex with the p50/p65 subunits of NF-kappa B when the NF-kappa B enhancer sequence from the Ig kappa light chain, rather than enhancer A, is used as probe; and both actions mimic the action of a phorbol ester. This suggests that iodide may regulate complex formation with NF-kappa B regulatory elements on multiple genes associated with growth and function, providing a potential mechanism relating the autoregulatory action of iodide on thyroid cells and its action on class I gene expression.

Animals↗
PubMed1997

Glycosylation variants of human TSH selectively activate signal transduction pathways.

The oligosaccharide chains of pituitary glycoprotein hormones such as human thyroid-stimulating hormone (hTSH) have been shown to be important in biosynthesis, subunit association, secretion and bioactivity. However, the exact biological significance of these glycosylation variants (isoforms) remains controversial. The aim of this paper is to investigate the role of hTSH glycosylation variants in signal transduction. Human pituitary standard TSH (2nd International Reference Preparation 80/558; IRP-hTSH) was treated with neuraminidase, fractionated by isoelectric focusing (IEF) and affinity chromatography using the lectins concanavalin A (Con A) and lentil. To determine the in vitro bioactivity of these hTSH isoforms, simultaneous measurement of cAMP formation and inositol phosphates release was applied in two different cell systems (CHO cells stably and Cos-7 cells transiently transfected with hTSHR cDNA). Desialylated TSH variants showed a significantly increased ratio of bioactivity to immunoreactivity for cAMP production in CHO-R cells (B/I ratio desialylated variants: 3.54 +/- 0.005; B/I ratio sialylated variants: 2.84 +/- 0.01 P < 0.05). Testing the bioactivity of hTSH glycosylation variants isolated by IEF, we found basic variants to be significantly more active than acidic ones in stimulating the cAMP formation in CHO-R cells (B/I ratio basic variants: 9.92 +/- 0.64; neutral variants: 5.98 +/- 0.07; acidic variants: 2.80 +/- 0.12; P < 0.01). There were no differences in stimulation of IP-release. High-mannose TSH variants (firmly bound to Con A) showed greater potency to stimulate cAMP formation and IP-release in both CHO-R and Cos-7 cells than biantennary TSH variants (weakly bound to Con A). Both core-fucosylated (lentil-bound) and core-unfucosylated (lentil-unbound) TSH variants proved to be strong stimulators of cAMP release in CHO and Cos-7 cells. In CHO-R (Cos-7) cells, 400 microU/ml core-fucosylated TSH stimulated cAMP formation 14(2.6)-fold, core-unfucosylated TSH 7.3(2.3)-fold over control values. In contrast to our findings of cAMP activation by both core-fucosylated and core-unfucosylated TSH variants, release of IPs was stimulated only by, core-fucosylated (lentil-bound) TSH variants and not by TSH variants lacking core-fucose residues (lentil-unbound TSH). This was true for both CHO-R and Cos-7 cells. The lentil-unbound TSH therefore showed an identical differential activation of signal transduction pathways in two different cell systems: strong stimulation of the cAMP-cascade without activation of IPs release (P < 0.05). In conclusion, we showed for the first time for TSH that the two dominant intracellular signal transduction systems (cAMP formation and IPs release) are activated to different degrees by hTSH glycosylation variants.

Animals↗
PubMed1997

Regulation of major histocompatibility complex class I gene expression in thyroid cells. Role of the cAMP response element-like sequence.

The major histocompatibility complex (MHC) class I gene cAMP response element (CRE)-like site, -107 to -100 base pairs, is a critical component of a previously unrecognized silencer, -127 to -90 bp, important for thyrotropin (TSH)/cAMP-mediated repression in thyrocytes. TSH/cAMP induced-silencer activity is associated with the formation of novel complexes with the 38-base pair silencer, whose appearance requires the CRE and involves ubiquitous and thyroid-specific proteins as follows: the CRE-binding protein, a Y-box protein termed thyrotropin receptor (TSHR) suppressor element protein-1 (TSEP-1); thyroid transcription factor-1 (TTF-1); and Pax-8. TTF-1 is an enhancer of class I promoter activity; Pax-8 and TSEP-1 are suppressors. TSH/cAMP decreases TTF-1 complex formation with the silencer, thereby decreasing maximal class I expression; TSH/cAMP enhance TSEP-1 and Pax-8 complex formation in association with their repressive actions. Oligonucleotides that bind TSEP-1, not Pax-8, prevent formation of the TSH/cAMP-induced complexes associated with TSH-induced class I suppression, i.e. TSEP-1 appears to be the dominant repressor factor associated with TSH/cAMP-decreased class I activity and formation of the novel complexes. TSEP-1, TTF-1, and/or Pax-8 are involved in TSH/cAMP-induced negative regulation of the TSH receptor gene in thyrocytes, suppression of MHC class II, and up-regulation of thyroglobulin. TSH/cAMP coordinate regulation of common transcription factors may, therefore, be the basis for self-tolerance and the absence of autoimmunity in the face of TSHR-mediated increases in gene products that are important for thyroid growth and function but are able to act as autoantigens.

Animals↗
PubMed1997

Geranylgeranylated rho small GTPase(s) are essential for the degradation of p27Kip1 and facilitate the progression from G1 to S phase in growth-stimulated rat FRTL-5 cells.

Cyclin-dependent kinase (Cdk) enzymes are activated for entry into the S phase of the cell cycle. Elimination of Cdk inhibitor protein p27Kip1 during the G1 to S phase is required for the activation process. An inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase prevents its elimination and leads to G1 arrest. Mevalonate and its metabolite, geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate, restore the inhibitory effect of pravastatin on the degradation of p27 and allow Cdk2 activation. By the addition of geranylgeranyl pyrophosphate, Rho small GTPase(s) are geranylgeranylated and translocated to membranes during G1/S progression. The restoring effect of geranylgeranyl pyrophosphate is abolished with botulinum C3 exoenzyme, which specifically inactivates Rho. These results indicate (i) among mevalonate metabolites, geranylgeranyl pyrophosphate is absolutely required for the elimination of p27 followed by Cdk2 activation; (ii) geranylgeranylated Rho small GTPase(s) promote the degradation of p27 during G1/S transition in FRTL-5 cells.

Animals↗
PubMed1997

Subtypes of anti-TSH receptor antibodies classified by various assays using CHO cells expressing wild-type or chimeric human TSH receptor.

To analyze the heterogeneity of anti-TSH receptor antibodies (TSHRAb), we measured serum TSH-binding inhibitory immunoglobulin (TBII), thyroid-stimulating antibody (TSAb), and thyroid stimulation blocking antibody (TSBAb) activities in 31 patients with positive TSHRAb, using CHO cells expressing wild-type TSHR (WT) or TSHR chimera (Mc2) wherein residues 90-165 were substituted by the LH/CG receptor. Using membranes from WT cells, we detected TBII activity in all 31 patients; 10 (32%), all with TSAb activity only, completely lost TBII activity using Mc2 membranes. TSAb activity was found in 26 sera using WT cells; 20 (77%) completely lost TSAb activity in Mc2 cells. Comparisons of TBII and TSAb activity in WT cells did not exhibit a strong positive correlation (r = 0.52). Of the 20 sera that completely lost TSAb activity in Mc2 cells, 10 retained some TBII activity in Mc2 cells. In each of the sera with retained TBII activity, TSAb activity was recovered in Mc2 cells using the conversion assay, which measures the conversion of a nonstimulating TSHRAb to a TSAb by the action of an anti-human IgG. Additionally, the TBII and conversion assay values in Mc2 cells exhibited a strong positive correlation (r = 0.86). Of the 31 sera, TSBAb was found in 7 samples, with no difference in WT and Mc2 cells. TBII activity was detected in all 7 sera with WT cells; TSAb activity in only 2. In the 5 sera with TSBAb but no TSAb activity, and with only a minimal or no decrease in TBII activity in Mc2 cell membranes, the in vitro conversion assay uncovered TSAb activity. Analyzing these data, we classify the sera into 5 groups containing multiple, different TSHR autoantibodies, including two different TSAbs, three different TBIIs, and one nonfunctional antibody. The heterogeneity of TBIIs as well as TSAbs provides a basis to explain the lack of correlation between TBII and TSAb activities in some past studies of Graves' sera.

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